UNCORRECTED PROOF Steroids xxx (2004) xxx–xxx A novel enzyme-linked immunosorbent assay for ethynylestradiol using a long-chain biotinylated EE2 derivative 3 4 Christian Schneider a , Heinz F. Schöler a , Rudolf J. Schneider b,∗ 5 a Institute of Environmental Geochemistry, University of Heidelberg, Im Neuenheimer Feld 236, D-69120 Heidelberg, Germany 6 b Institute of Plant Nutrition, University of Bonn, Karlrobert-Kreiten-Str. 13, D-53115 Bonn, Germany 7 Received 7 January 2003; received in revised form 29 December 2003; accepted 26 January 2004 8 9 Abstract 10 Ethynylestradiol (EE2) is one of the most potent endocrine disrupting compounds capable to induce estrogenic effects even at trace level concentrations in the aquatic environment. Methods for detecting EE2 in such concentrations are generally based on GC or HPLC coupled to at least one mass spectrometer. Another approach are immunoassays and sensor systems but for most designs, derivatives of EE2 are required (e.g. for coupling to carrier proteins, enzyme or fluorescent labels, etc.). Here we present the straightforward synthesis and complete characterization of a new long-chain biotinylated EE2 derivative. The new EE2 derivative is used as tracer in a direct competitive enzyme-linked immunosorbent assay (ELISA) for the determination of EE2. With pure water, the limit of detection (LOD, signal-to-noise ratio, S/N = 3) and the test midpoint were found to be 14 and 136 ng l -1 , respectively. Cross reactivity (CR) was tested for 10 endogenous steroids and the BSA-conjugate used for immunization, as well as a synthetic precursor of the conjugate. Among the naturally occuring compounds CR was determined to be maximum for metabolites of EE2 conjugated at ring-position 3 (17% and 37% for 3-glucuronide and 3-sulphate, respectively). Assay stability was tested against humic substances and organic solvents. Increasing amounts of organic solvents in the sample caused a clear decrease in sensitivity, presence of humic substances lead to an overestimation of EE2. 11 12 13 14 15 16 17 18 19 20 21 © 2004 Published by Elsevier Inc. 22 Keywords: Ethynylestradiol; EE2; Biotinylation; Steroid; Biotin; Synthesis; ELISA 23 24 1. Introduction 25 The 1990s have witnessed a great expansion of interest 26 in the implications of environmental exposure of wildlife 27 and humans to potential endocrine disruptors [1]. Gutendorf 28 and Westendorf [2] identified more than 50 compounds 29 exhibiting endocrine disrupting effects, with the majority 30 of these compounds being of estrogenic nature. Estradiol 31 and its metabolites estrone and estriol have been detected 32 in Europe [3–6], North America [6] and Asia [7] in munic- 33 ipal and industrial effluents. The pharmaceutical estrogen 34 17-ethynylestradiol (EE2) frequently prescribed as an oral 35 contraceptive has been detected at trace-level concentrations 36 in the low ng l -1 -range in sewage effluents in Italy, Ger- 37 many, England, The Netherlands and the US [3,4,6,8–13]. 38 In surface waters EE2 was only detected in a few samples. 39 Some studies reported values up to 4.3 ng l -1 [3]. How- 40 ever, most of the samples had levels below 1ppt [3,8,12]. 41 ∗ Corresponding author. Tel.: +49-228-732856; fax: +49-228-732489. E-mail address: schneider@uni-bonn.de (R.J. Schneider). Even though the detected EE2 concentrations are very low 42 there might still be an impact on the aquatic ecosystem, as 43 has been indicated by laboratory studies using vitellogenin 44 as biomarker for endocrine disruption in rainbow trout 45 [14]. 46 For quantifications in the low ng l -1 -range analysts 47 rely on highly sophisticated methods such as GC–MS, 48 HPLC–MS or even tandem MS techniques. As an alterna- 49 tive, sensor techniques and immunochemical assays such 50 as enzyme-linked immunosorbent assay (ELISA) can be 51 used. In the past, several studies have been published us- 52 ing immunoassays for measuring EE2 in different matrices 53 [15–19]. Available test kits for EE2 are optimized for bi- 54 ological specimens such as blood and urine. Applicability 55 to environmental samples was neither considered nor as- 56 sessed for the majority of these kits. Here we present the 57 facile synthesis and complete characterization of a new 58 long-chain biotinylated EE2 derivative. We have employed 59 an ε-aminocaproic acid spacer connecting the biotin moi- 60 ety to an EE2-6-carboxymethyloxime with the intention 61 of maximizing the biotin–avidin interaction [20,21] in the 62 1 0039-128X/$ – see front matter © 2004 Published by Elsevier Inc. 2 doi:10.1016/j.steroids.2004.01.003 STE 5928 1–9